lnt

J. Rodration

Oncology Biol. Pkys..

1976. Vol.

I. pp. 45.5-W

Pergamon Prec5.

Printed m the L’.S.A

PHYTOHEMAGGLUTININ-STIMULATED LYMPHOCYTES: DNA SEDIMENTATION

HUMAN BEHAVIOR?

NADINE TIMBERLAKE,Ph.D.,SAMUEL HELLMAN, M.D. and JAMES A. BELLI, M.D. Joint Center for Radiation Therapy, Department of Radiation

Therapy,

Harvard

Medical

School. Boston, MA 0211S. U.S.A. The sedimentation behavior of DNA from mitogen-stimulated human peripheral lymphocytes in alkaline sucrose density gradients suggests a unique intranuclear conformational organization. This is expressed by rapid denaturation of DNA at high pH under conditions of extraction which otherwise yield large, duplex DNA molecules. The data further suggest that lymphocytes contain DNA which lacks tertiary structure typical of chromation in other tissue cells. X-irradiation produced measurable damage in lymphocyte DNA and this damage was repaired, but at a much reduced rate. DNA repair was related to the cell cycle and it was found that pulse labelled lymphocvtes accomplished repair more efficiently during DNA synthesis. Lymphocyte radiosensitiv&y may be related to DNA conformational factors rather than the absence of repair processes present in other mammalian cells. Human lymphocytes,

DNA, Phytohemagglutinin.

INTRODUCTION

The small, mature, circulating lymphocyte expresses radiation injury by intermitotic death following small X-ray doses; the postirradiation intracellular events responsible for this response are unclear despite substantial work devoted to this question.‘-“.‘“~I.~” Among the possible explanations for the radiosensitivity of mature lymphocytes may be that these cells have impaired or absent capacity for repairing radiation damage in DNA because such repair capacity may be related closely to whether or not a cell is “in cycle”. This is a reasonable possibility because there is evidence that lymphocytes stimulated to divide (“blast transformation”) by various plant mitogens, e.g. phytohemagglutinin, demonstrate decreased radiosensitivity, relative to the unstimulated lymphocyte. with regard to a number of end-points.2h.‘T-32 However, there is evidence that non-dividing lymphocytes are capable of molecular repair processes following various levels of damage,*.u lO.ii-,9.24.?4 1 Sedimentation

behavior

of mammalian

cell

‘Supported by USPHS research grants CA12662 and CA-10941 and training grant CA-05137

DNA in alkaline sucrose density gradients has been used to study the production of X-ray induced single-strand breaks and their rejoining during the immediate post-irradiation period. 1,8.12.1.)6.20.22.37 The non-dividing lymphocyte is difficult to study in this way because of the absence of DNA synthesis, necessitating non-radioactive isotopic identification of DNA after sedimentation. Scaife”’ examined the sedimentation pattern of DNA from irradiated, non-dividing thymocytes with spectrophotometric techniques and found repair of DNA damage as measured by molecular weight changes. Similar results were reported by Karran and Omerod’” in murine splenic and thymic lymphocytes using flurometric methods for assaying DNA following sedimentation in alkaline sucrose gradients. Lymphocytes stimulated to undergo cell division have also been reported to repair strand breaks in their DNA as manifested by changes in alkaline sucrose sedimentation behavior. Huang et al.” reported that moderate doses of X-irradiation (5 kR) reduced DNA molecular weight by approximately from the National Institutes of Health.

45’

Cancer

Institute,

National

456

Radiation Oncology 0 Biology 0 Physics

March-April

one-half, with return to the pre-radiation molecular weight following a 60-min incubation period. Donlon and Norman’ studied the kinetics of rejoining of single-strand breaks in phytohemagglutinin-A (PHA) - stimulated human lymphocytes, and found an initial period of rapid repair characterized by a rejoining half-time of approximately 15 min which was essentially completed within 40 min. This was followed by a “longer term” process during which the rate of change in strand break disappearance decreased. In view of the importance of peripheral lymphocytes in cell mediated immune responses as these may relate to neoplastic disease, and because modification of these responses may occur during the radiotherapeutic management of cancer, we initiated studies to clarify those factors responsible for the unusual radiosensitivity of this cell type. METHODS

AND MATERIALS

I. Cell culture and DNA labelling A flow diagram of the experimental design is presented in Fig. 1. Whole blood, drawn into vacutainer tubes containing EDTA, was collected from healthy, human volunteers, and mixed in 50 ml plastic disposable syringes with Lymphocyte Separating Reagent (Technicon) containing carbonyl-iron and dextran at a ratio of 1:4. After mixing at 37°C for 30 min, the cell suspension was allowed to settle for 1 hr and the supematant passed around a magnet through sterile transfer tubes (Technicon Lymphocyte Separator) to remove the phagocytes containing carbonyl-iron yielding a preparation containing 297% lymphocytes. The cell suspension was centrifuged at 300g for 15 min at room temperature and the supemant discarded. The pellet was resus-

1976. Vol. 1, Number 5 and Number 6

pended in growth medium (EM-15) consisting of Eagle’s minimal-essential medium supplemented with glutamine, penicillin, streptomycin, heat inactivated fetal calf serum (15% volume/volume (v/v)) and 40 units/ml of preservative-free heparin and adjusted to a concentration of 1 x 10”lymphocytes/ml. Oneml samples were cultured in 12 x 75 mm disposable plastic culture tubes (Falcon). Reconstituted bacto-phytohemagglutin M (PHA) was added to each tube to a final concentration of 50 kg/ml. Cultures were then allowed to incubate from 40 to 92 hr at 37.5”C in a humidified atmosphere of 97% air-3% COZ. PHA-stimulated lymphocytes were incubated in thymidine-methyl-‘H (6.7 Ci/mM, 0.5 &i/ml) in EM-15 for 20 hr (70-92 hr in culture for continuous, uniform labelling of DNA), or for 0.5 hr (39-5-40 hr in culture, 6.7 Ci/mM, 20 &i/ml) providing labelling of newly synthesized DNA. When lymphocytes were incubated after continuous or pulselabelling, the medium was further supplemented with non-radioactive thymidine (0.46 pglml). II. Autoradiography Labelled cells were prepared for autoradiography by incubating them at room temperature (23°C) for 7 min in Puck’s Saline A containing 0.25% trypsin and 10-l M EDTA. At the end of this period, 0.3 ml centrimid (Fisher) was added; the erythrocyte-free preparation was spun for 2 min at 16OOg,the supematant discarded, and 2 ml acetic acid (50%) carefully added. The acetic acid was decanted immediately and replaced with one drop of filtered aceto-orcein. Prepared slides were (1) dipped into warmed (46°C) Ilford G-5 emulsion

Collect,

separate

culture

L

0 HOURS

IN

Cl/L?-URE

Fig. 1. Flow diagram of experiment design. The arrows indicate the time of irradiation for pulse labelled cells. X-irradiation of continuously labelled cells was always at the end of the labeiled period (92 hr).

Human

lymphocyte

DNA

sedimentation

for 3 set; (2) allowed to dry for 3-4 hr; (3) sealed in light-proof boxes and stored in the refrigerator until ready for developing; and (4) examined microscopically to determine the labelling index. III. irradiation Each l-ml culture sample was dispersed evenly in a 100 x 15 mm plastic petri dish (Falcon) which was placed without its cover in a holder3 and irradiated in room air at 0-4”C. X-radiation was produced in an OEG-60 Machlett beryllium end-window tube operated at 50 kVp and 20 mA. With 0.06 mm Al added filtration, the beam had a half-value layer of 0.12 mm Al. At a distance of 24 cm from the window to cell-surface interface, the dose-rate was 1278 R min-‘.? Following irradiation, the cells were transferred to new culture tubes, and either kept in ice or placed at 37.5”C for varying intervals.

0 N. TIMBERLAKE

et al.

457

displaced from the top of the gradient onto 2.1 cm glass fiber filter discs. They were dried and placed in scintillation vials with 10 ml scintillation fluid consisting of 4 g OMNIFLUOR (New England Nuclear) in one liter toluene. Radioactivity was assayed in a Beckman LS-233 scintillation spectrometer operated at ambient temperature. Results are expressed as per cent total radioactivity recovered from each gradient. Recovery was found to be close to 100% of that amount layered initially, usually 5000-10,000 counts per minute (cpm).

IV. Alkaline sucrose gradient centrifugation Velocity-sedimentation patterns of lymphocyte DNA were obtained using techniques described by McGrath and Williams*’ as modified by Elkind and Kamper.* Preformed linear alkaline sucrose gradients of 4.8 ml volume were prepared in 5 ml polyallomer centrifuge tubes. A 5 per cent sucrose weight/ volume (w/v) solution contained: O-8 M NaCl, 0.2 M NaOH and 0.003 M Na2EDTA (pH = 124-12.9); 20% sucrose (W/V): O-6 M NaCl, O-4 M NaOH and O-003 M Na*EDTA (pH = 12.3-12.7). At the time of use, the gradients were carefully overlayed with 0.25 ml lysing solution consisting of O-4 M NaCl, 0.05 M NaOH and 0.01 M Na2EDTA (pH = 12.9). Cell samples, ranging in volume from 0.05 to 0.1 ml were introduced gently into the lysing solution and stored at room temperature for 60 min in the absence of visible light. Following this lytic storage period, the tubes were spun at 36,OOOrpm using a SW 50.1 swinging bucket rotor in an L3-50 (Beckman) preparative ultracentrifuge for 1 hr at approximately 12°C. Using 50% sucrose solution, 30-ten drop fractions were

RESULTS Under our culture conditions, approximately 60% of stimulated lymphocytes underwent transformation as measured by morphologic criteria. Of this transformed population, 85% (50% of the total population) incorporated ‘HTdR over a 20 hr interval 72 to 92 hr in culture as shown by radioautographs. Thus, while the DNA sedimentation data presented below represent the behavior of DNA from only 50% of the total lymphocyte population cultured, these data are clearly relevant to those lymphocytes capable of responding to PHA-stimulation. Under our conditions of extraction and sedimentation, DNA from unirradiated Chinese hamster cells (V79-753), exhibit sedimentation profiles in alkaline sucrose gradients which are bimodal; the general features of this behavior are illustrated in Fig. 2, panel A and are consistent with those reported by Elkind and Kampe? and Elkind’ who designated the sharp peak of radioactivity near the meniscus as “complex” and the broader, less prominent peak near the middle of the gradient as “main peak”. We refer to these as peaks I and II for reasons discussed in a recent communication from this laboratory.33 In brief, we reported that peak I DNA from V79-753 cells was entirely duplex while that contained in peak II was a mixture of duplex and single-stranded material. In contrast, the sedimentation profile derived with DNA from PHA-stimulated lym-

tAl1 X-ray doses are expressed as exposure doses (Roentgens) because of the uncertainty of the

conversion factor to rad for this beam energy and quality.

158

Radiation Oncology ??Biology 0 Physics

March-April

1976, Vol. 1, Number 5 and Number 6

SEDIMENTATION

I v79-753

OR / HUMAN

PERIPHERAL

OR

Y-l

Fig. 2. Sedimentation of DNA from Chinese hamster cells (panel A) or stimulated lymphocytes (panel B). In this, and in subsequent Figures, sedimentation is from left to right. The prominent peak in panel A at fraction 8 is peak I and that at fraction 13, peak II. Lymphocyte DNA consists entirely of peak II material. From: Belli, J. A., Timber&&e, N. Hellman,S.:BrookhavenNatlLab.Publ.No.50418,pp.207-217,1974. phocytes (Fig. 2, panel B) was very close to Gaussian in distribution and the peak of radioactivity in the gradient corresponded to that portion of the hamster cell DNA profile designated peak II. Decreasing the lytic storage time to 0.25-0.5 hr (the shortest times possible in our system) did not result in bidmodal sedimentation profile; the patterns remained Gaussian and not different than that shown in Fig. 2, panel B. We take these results to mean that DNA from the PHAstimulated lymphocyte has an intranuclear conformational organization which permits more rapid denaturation at high pH. We tested the hypothesis that stimulated lymphocyte DNA exists at a conformational organization that permits more rapid denaturation at high pH by treating lymphocytes with nitrogen mustard (HN2) before lysis. Because of its cross-linking properties (DNADNA and/or DNA-protein), this agent stabilized duplex DNA at high PH.‘~ Figure 3 shows the resultant profile after lymphocytes were treated with HN2, 1.0 kg/ml, for 30 min. The sedimentation behavior closely resembled that obtained with unirradiated Chinese hamster cell DNA, i.e. peak I (duplex) and peak II (duplex and single-stranded). This constitutes strong evidence that the sedimentation properties of DNA from the unirradiated stimu-

SEDIMENTATION

b

/ ugm/m/ ,30 min

37

oc

HIV2

42 %

J

II

J

Fig. 3. Effect of nitrogen mustard on lymphocyte DNA sedimentation. The closed circles trace the profile after HN2 treatment and should be compared to that obtained with V79-753 cell DNA (Fig. 2, panel A). From: (see Fig. 2). lated lymphocyte reflect unique organizational rather than size characteristics. Of the several factors which promote resolution of peak I DNA into peak II. X-radiation has been studied extensively. Following 2OOOR, Chinese hamster cell DNA exhibits sedimentation behavior illustrated in

Human lymphocyte DNA sedimentation 0 N.

Fig. 4, panel A; only peak II DNA was observed which is consistent with similar effects observed by others.‘.‘.‘.’ DNA from PHA-stimulated lymphocytes irradiated with the same radiation dose exhibited sedimentation behavior (Fig. 4, panel B) which reflected the presence of a significant amount of slower sedimenting DNA species; this finding was taken to be evidence of the production and expression of radiation induced strand damage. Approximately 7000-8000 R would have been required to achieve the same sedimentation profile with V79-753 cell DNA. “Repair” of “single-strand breaks” in mammalian cell DNA is presumed when its post-irradiation behavior returns to preirradiation patterns after irradiated cells are incubated for varying intervals following Xirradiation. Such effects have been noted for Chinese hamster cell DNA;2.7.s the dosedependent damage expression has been found to be: peak I + peak II + single strand breaks. If sufficient time is allowed after irradiation, Chinese hamster cells reverse this damage sequence which leads to restoration of alSEDIMENTATION

,A I5C

vrs-753

p22

I

&q

-

~2000R

---2000R+ZHr

4

z $

---

-

ZOOOR OR

r\

n

TIMBERLAKE

459

et al.

kaline stability of duplex DNA.’ This damagerepair sequence is illustrated in Fig. 4, panel A. PHA-stimulated lymphocytes also demonstrated repair-capacity. However, the process required longer times and the data suggested incomplete repair. In Fig. 4, panel C, we show that after 2000 R followed by O-5 hr at 37°C little change in the sedimentation profile was apparent (compare with panel B). It has been shown in a variety of mammalian cells that 0.5 hr is sufficient for the repair of most, if not radiation induced strand DNA all, breaks.?.%8.3%)7An incubation period of two hours was required after exposure for lymphocyte DNA to demonstrate sedimentation behavior which closely approximated that from unit-radiated cells (Fig. 4, panel D). Figure 5, panels A and B illustrate the sedimentation profiles of DNA from V79-753 cells (panel A) and lymphocytes (panel B) after exposure to 1OkR. In panel B, the dashed line traces the sedimentation of DNA from unirradiated lymphocytes. It is clear (1) that smaller DNA molecular species emerge after 10 kR than after 2 kR (compare with Fig. 4, panel B) and (2) that 10 kR apparently produced a greater level of damage in lymphocytes than in V79-753 cells (panel A, closed circles). In the former, the peak position was fraction 8; in the latter, fraction Il. These observations suggest that more damage is SEDIMENTATION .

; C

-2cmR+0sHr --OR

D

-ZOOOR+ZHr --

OR

15-

r, I,

---

!0,000R l0,000R+2H1

I/~ c 15-

FRACTION

v79-753(

A

B

-l0,000R ---OR

_

Jq&f

--.10,000A+05~r ---OR

I 0 c

C--410,000R+ ---OR

NUMBER

Fig. 4. Effect of 2000R on DNA sedimentation. Panel A, V79-753 after X-ray (0) and following 2 hr repair time at 37.5”C(---). The latter is the same as the profile in Fig. 2, panel A. Panels B, C and D trace the DNA sedimentation profile for lymphocytes after 2000R (panel B) followed by 0.5 hr (panel C) or 2 hr (panel D) repair times. The dashed line in each is the profile for unirradiated lymphocytes.

FRACTION

NUMBER

Fig. 5. As Fig. 4. but for 10 kR.

enr

460

Radiation Oncology 0 Biology 0 Physics

expressed by lymphocyte DNA in alkaline sucrose gradients compared to V79-753 cells reflecting, as discussed below, differences in intranuclear organization rather than smaller energy requirements to produce alkali labile regions in individual DNA strands. Following 10 kR, a radiation level which produced substantial DNA damage in both hamster cells and stimulated lymphocytes (Fig. 5, panels A and B), repair of DNA, as measured by post-irradiation sedimentation behavior was relatively complete in hamster cells (Fig. 5, panel A, dashed line) as demonstrated by the appearance of peak I and peak II DNA 2 hr after exposure; repair in lymphocyte DNA was less complete (Fig. 5 panels C and D). To study the effect of cell cycle position on DNA repair, we utilized pulse labelling techniques followed by “chase” in the presence of non-radioactive thymidine for varying intervals. Stimulated lymphocytes were exposed to ‘HTdR for 0.5 hr as described above (see Fig. 1) from 39.5 to 40-O in culture. This approach allowed examination of only that portion of the total DNA synthesized by those cells in S during the pulse and its behavior as progression into postsynthetic cell cycle compartments occurred during “chase” periods. In Fig. 6 we show the sedimentation patterns of lymphocyte DNA at times after

March-April

1976, Vol. 1, Number 5 and Number 6

pulse labelling. A broad distribution of DNA sizes was observed immediately after a 0.5 hr pulse; most of the material sedimented close to the meniscus (panel A). As “chase” time increased to 6 hr, larger DNA species were observed indicating incorporation of newly synthesized DNA segments into larger single strands (panels B-F). The nature of the low molecular weight DNA between fractions four to seven remains obscure and is not considered in this report. At any rate, it constituted a small fraction of the DNA in the gradient and was an inconsistent finding. To distinguish repair of radiation damage and continued DNA synthesis (both leading to larger molecular sizes), we used hydroxyurea (2 mM) to inhibit DNA synthesis. This drug apparently does not affect single-strand break rejoining.23 Figure 7 shows the results observed when lymphocytes were pulse-labelled as described above. Pulse labelled cells were then incubated in 2 mh4 hydroxyurea (panels A and B). In the presence of this drug, growth of the labelled strand was inhibited over a 2 hr interval relative to untreated control cells (panel B). Following 2000 R, exposure to hydroxyurea immediately after irradiation did not influence the expression of damage (panel C); continued exposure to hydroxyurea for 2 hr yielded a sedimentation profile similar to that for unirradiated cells similarly treated

SEDIMENTATION

Fig. 6. Change in lymphocyte DNA sedimentation for various chase periods after pulse labelling. The increase in heavier DNA species is evidenced by the change in peak fraction toward the right.

Human lymphocyte DNA sedimentation 0 N. TIMBERLAKE et al.

SEDIMENTATION

IO-

-HL

*-

0

10

20

30

0

IO

FRACTION

20

30

NUM8ER

Fig. 7. Effect of hydroxyurea on DNA synthesis and strand repair after 2ooO R, pulse labelled cells. Panels A and B show data with unirradiated cells; strand growth is inhibited, panel B, closed circles. The presence of hydroxyurea (HU) during irradiation did not influence the sedimentation pattern (panel C). Two hr after 2000R in the presence of HU (0, panel D) did not result in repair inhibition (compare with 0, panel B).

with hydroxyurea. Thus, repair of DNA strand damage proceeded in the face of decreased DNA synthesis. Figure 8 shows the results we observed when cells were incubated for 3 hr after labelling and before X-irradiation. This “chase” interval was long enough to allow near completion of strand growth, but short

461

enough so that most of the labelled cells were in late-S. Following 10 kR, significant damage was expressed in the form of slower sedimenting species (panel A). Panels B, C and D show profiles after repair times of 0.5, 2 and 4 hr respectively; most of the radiation damage registered was repaired by 2 hr. However, it is important to note that significant amounts of low molecular weight material remained after a 0.5 hr repair time (note the bulge on the trailing edge, panel B). A 9 hr “chase” period was used to study the DNA repair capacity of non-S cells (Fig. 9). Comparable damage was expressed following 10 kR (panel A), but repair to preirradiation sedimentation behavior did not occur until 4 hr after irradiation (compare with Fig. 8). Substantial levels of damage, as expressed by slower sedimenting material, remained after repair periods of O-5 and 2 hr (panels B and C; compare with similar post-irradiation incubation periods in Fig. 8). In addition, we take the relatively small peak near the meniscus in the control protile in Figs. 8 and 9 to indicate small molecular-size fragments liberated apart from those induced by radiation damage. The trailing bulge in panel C (Fig. 9), was not observed for a comparable repair time (2 hr) for S cells (panel C, Fig. 8), and very likely represents DNA fragments not present to begin with and not repaired by non-S cells. Thus, non-S cells appeared to

SEDIMENTATION A

-10.000*

-10.oooR

8

r05Hr

SEDIMENTATION

~A

u

-

C

Y

-

IO.OOOR +

0

2 H,

-IO,owRr4H,

---OR

---

n

IO-

5-jJL 0

IO

20

0

FRACTlOn;

to

/ ,

8

1

-IaOOOR+05kr ---OR

I

OR

JqTL 30

u10,oom ---OR

20

~ 30

h’UMBER

Fig. 8. Effect of 10 kR on pulsed labelled lymphocytes after 3 hr chase period. Sedimentation pattern returns to pre-irradiation appearance by 2 hr. The profile traced by the dished line (OR) is repeated in each panel for reference and is taken from data for pulse-chase experiments similar to those in Fig. 6.

FRACTION

NUMBER

Fig. 9. As Fig. 8, except that chase period was 9 hr. Profiles do not return to pre-irradiation appearance for up to 4 hr after 10 kR. The OR profile (---) is taken from Fig. 6, panel F.

462

Radiation Oncology 0 Biology 0 Physics

require a longer time than S cells to restore profile to its pre-irradiation pattern. DISCUSSION The sedimentation behavior of lymphocyte DNA can be examined in the light of previous work in our laboratory’3 which clarified the molecular nature of mammalian cell DNA in alkaline sucrose gradients. The essential features were that DNA extracted from cells at high pH and sedimented through a sucrose gradient was predominently duplex DNA. A smaller fraction of the material consisted of duplex and single-stranded molecules. We have designated these as peaks I and II respectively and correspond to “complex” (peak I) and “main peak” (peak II) DNA originaIly described by Elkind and Kamper.’ Interest in peak I DNA centers about its apparent anomalous sedimentation properties;‘.’ its resolution into peak II DNA by small radiation doses’.‘.7.8 and actinomycin D;’ its ease of resolution by lysis at high temperatures;‘4.33 its increased stability following treatment with cross-linking agents;‘3 and its restoration with time after .X-irradiation.2.7.8 These observations constitute strong evidence that the sequential liberation of DNA molecules of unexpected conformation (duplex at high pH) is a consequence of the intranuclear organization of nucleoprotein and/or chromatin and not an artifact of technique that should be avoided if possibie (see Ref. 33 for further discussion on this point). DNA from mitogen-stimulated lymphocytes demonstrated unique sedimentation properties in alkaline sucrose density gradients. (1) Lymphocyte DNA, compared to that liberated from V79 Chinese hamster cells, exhibited lability relative to strand separation at high pH. This observation suggests that mitogen stimulation of small lymphocytes, while resulting in transformation to a dividing cell, does not promote those organizational relationships between DNA and non-nucleic acid material which are necessary to render nucleoprotein resistant to alkali denaturation. It is not unreasonable to expect that mitogenstimulated lymphocytes may have functional limitations compared to other tissue cells

March-April

1976, Vol. 1. Number 5 and Number 6

which contain nucleoprotein in a more stable organizational and, possibly, functional condition. In addition, Simpson ef al.” have suggested that the alkali-resistance of Chinese hamster DNA may be due to natural crosslinks between DNA and DNA or DNA protein. Such cross-links would be expected to impose tertiary structure on DNA helices which result in decreased denaturability at high pH. DNA in the stimulated lymphocyte niay lack these cross-links; such cross-links can be provided artificially by treating lymphocytes with nitrogen mustard, and following such treatment, lymphocyte DNA exhibits sedimentation behavior which strongly suggests inefficient strand separation (see Fig. 3). (2) When exposed to moderate levels of X-radiation, DNA from stimulated lymphocytes exhibits sedimentation properties characteristic for the presence of significant numbers of single strand breaks (Fig. 4). The same dose of radiation to Chinese hamster cells results only in the resolution of peaks I to II DNA. It is attractive to consider these differences in DNA sedimentation as the basis for the increased radiosensitivity of lymphocytes compared to other cells. However, the resolution of peak I to peak II (double stranded to predominently single-stranded material) may be the consequence of radiation induced single strand breaks which then promote duplex unwinding. Thus, while both cell types may experience the same level of damage by comparable radiation doses, lymphocyte DNA expressed more damage because its level of conformational organization differs significantly from Chinese hamster cells. Lymphocyte radiosensitivity may, therefore, be directly related to conformational states of DNA within the stimulated (and, by implication, the unstimulated) cell. (3) Stimulated lymphocytes repair radiation damage to their DNA as measured by return of the sedimentation profile to that comparable to pre-irradiation patterns. However, two observations should be noted. First, low molecular-weight material persists after a prolonged repair interval (2 hr); second, the rate of repair is significantly slower than that observed in Chinese hamster cells. In the latter, all single strand breaks are repaired

Human lymphocyte

DNA sedimentation

within 2&30 min after X-irradiation; lymphocytes require longer than 2 hr to accomplish significant, but incomplete repair. (4) By using pulse-labelled lymphocytes. thus restricting our observations to those cells in DNA synthesis at the time of the pulse, we found that strand repair after irradiation was most efficient during S (within O-3 hr after pulse). As cells progressed into non-S compartments of the cell cycle, increasing amounts of low molecular weight materials persisted after post-irradiation repair interval (Figs. 8 and 9). These data suggest that conformational organization of DNA at the cellular level, which is reflected by duplex stability at high pH, may not be crucial to repair capacity at least relative to strand rejoining. when DNA replication enzymes are active; the latter may serve an important function during strand repair processes. Thus. during DNA synthesis, conformational factors appear to have a less important influence on

?? N. TIMBERLAKEet al.

463

the repair capacity of irradiated lymphocytes. On the other hand, when lymphocytes are irradiated during non-S cell cycle compartments, organizational factors may again assume importance for the expression of strand repair because of the decreased function of replicative enzyme systems-systems which may accomplish strand rejoining with a sufficient degree of efficiency to mask the conformation influences otherwise present. Therefore, our findings suggest the presence of a unique DNA conformation in the stimulated lymphocyte. This conformation is expressed by rapid strand separation at high pH and may be a reflection of the absence of cross-links otherwise present in the DNA of other tissue cells. We suggest that this unique property of lymphocyte DNA may be one of the important factors responsible for the radiosensitivity of this cell type and may play a role in the ability to repair radiation-induced DNA damage.

REFERENCES 9. Evans, R.G., Norman, A.: Radiation-stimulated incorporation of thymidine into the DNA of tion properties of mammalian-cell DNA: Evidence that nonspecific aggregation does not human lymphocytes. Nature (Lo&.) 217: occur during cell lysis. Int. J. Radiat. Biol. 21: 455AS6, 1%8.

1. Belli, J.A., Cooper. S., Brown, J.A.: Sedimenta-

603-606, 1972. 2. Belli, J.A., Nagle, W.A.: Factors which influence the sedimentation properties of mammalian cell DNA: Effect of two-dose Xirradiation. Israel J. Chem. 10: 1241-1253, 1972. 3. Belli. J.A., Shelton, M.: Potentially lethal radiation damage: Repair by mammalian cells in culture. Science 165: 490-492. 1%9. 4. Belli, J.A., Timberlake, N., Hellman, S.: The effects of radiation on the lymphocyte response to nitrogens. Brookhaven Nat1 Laboratory Symposium on Interaction of Radiation and Host Immune Defense Mechanisms in Malignancy: Brookhaven Natl Lab.. No. 50418, 1974. pp. 207-217. 5. Connor, N.G., Norman, A.: Unscheduled DNA synthesis in human leucocytes. Mut. Res. 13: 393-402, 19?1. 6. Donlon. T., Norman, A.: Kinetics of rejoining of single-strand breaks induced by ionizing radiation in DNA of human lymphocytes. Mut. Res. 13: 97-107. 1971. 7. Elkind, M.M.: Sedimentation of DNA released from Chinese hamster cell. Biophys. J. 11: 502-520. 197 1. 8. Elkind. M.M.. Kamper, C.: Two forms of repair of DNA4 in mammalian cells following irradiation. Bioghgs. J. 10: 237-245, 1970.

10. Evans, R.G., Norman, A.: Unscheduled incorporation of thymidine in ultraviolet-irradiated human lymphocytes. Radiat. Res. 36: 287-298, 1%8. 11. Huang, A.T., Krener, W.B., Laszlo, J., Stelow, R.B.: DNA repair in human leukemic lymphocytes. Nature New Biol. 240: 114-115, 1973. 12. Humphrey. R.M., Steward, D.L., Sedita, B.A.: DNA-strand breaks and rejoining following exposure of synchronized Chinese hamster cells to ionizing radiation. Mut. Res. 6: 459-465, 1968. 13. Karran, P., Ormerod, M.G.: Is the ability to repair damage to DNA related to the proliferative capacity of a cell? The rejoining of X-ray-produced strand breaks. Biochem. Biophys. Acta 299: 54-64, 1973. 14. Kohn, H.I., Belli. J.A.: Duration and temperature of extraction change the sedimentation parameters of mammalian DNA measured in alkaline sucrose gradients. Radiat. Res. 55: 518, 1973 (abstract). 15. Lett, J.T., Caldwell, I., Dean, C.J.,.Alexander, P.: Rejoining of X-ray induced breaks in the DNA of leukaemia cells. Nature (Land.) 214: 790-792, 1967. 16. Lett, J.T., Sun, C.: The production of strand breaks in mammalian DNA by X-rays at

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different

stages in the cell cycle. Radial.

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44: 771-787, 1970.

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